title: Attenuation of 19-9 antigen secretion in human colorectal carcinoma cell cultures by transfection with cDNA encoding novel ADP-ribosylation factor-like proteins.
abstract: We have used cDNAs coding for novel ADP-ribosylation factor-like molecules (ARL184 and ARL184Delta) to alter 19-9 antigen glycoprotein secretion in cultured human colorectal carcinoma cells SW1116 by transfection and cloning. This ARL contains a lipophilic N-terminal with an isoleucyl and 3 leucyl residues, 4 functioning consensus sequence GTP binding sites, and 184 total aminoacyl residues. An ARL cDNA was also constructed deleting the codon for the N-terminal glycyl moiety. The resulting cell clones were shown by Northern blots to overexpress ARL mRNA. Electron microscopy-immunocytochemistry also indicated the overexpression of ARL granules subcellularly. Secretion of the tumor-associated 19-9 antigen into apical medium was decreased 3- to 5-fold and the secretion of TCA/PTA precipitable 3H-labeled glycoprotein was decreased by 34% in clone SW1116(ARL184)Delta. Western blot analyses of cell homogenates and media were in agreement with the secretion assays and showed a diminution of 170-200 kDa, 19-9, antigenicity in transfected cells and their media. Apical secretion of 19-9 antigen was diminished 14-fold in cells, SW1116 (ARL184)alpha, transfected with the complete ARL cDNA sequence, suggesting that the glycyl moiety may be required for maximal abatement. However, incorporation of label from [3H]myristate into 22-kDa bands of NP-40 extracts and ARL-antigenic molecules of parent cells was 3-fold greater than that in samples from the two transfectants; thus the transfected cells may not myristylate the overexpressed ARL efficiently. Notwithstanding the N-terminal glycyl moiety undergoing some other modification, we conclude that overexpression of this ARL is sufficient to generate a 19-9-deficient phenotype. These ARLs may eventually disrupt terminal oligosaccharide glycosylation, resulting in an apparent diminished exocytosis of 19-9 glycoprotein carriers by transfected and cloned cells.
authors.affiliation: Biochemistry of Cellular Transport Laboratory, CNRS, University of Paris XI, Building 432, Orsay Cedex, 91405, France.
authors.forename: D
authors.initials: D
authors.lastname: Eboue
authors.forename: C
authors.initials: C
authors.lastname: Icard-Liepkalns
authors.forename: T M
authors.initials: TM
authors.lastname: Beringer
authors.forename: V A
authors.initials: VA
authors.lastname: Liepkalns
mesh_terms: D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000032:analysis / Q000378:metabolism*; D015179:Colorectal Neoplasms / Q000276:immunology*; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes / Q000276:immunology; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006023:Glycoproteins / Q000032:analysis / Q000276:immunology; D006801:Humans; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009226:Myristates / Q000378:metabolism; D009363:Neoplasm Proteins / Q000032:analysis; D010168:Palmitates / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D014162:Transfection / Q000235:genetics; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D000951:Antigens, Neoplasm; D018076:DNA, Complementary; D000939:Epitopes; D006023:Glycoproteins; D009226:Myristates; D009363:Neoplasm Proteins; D010168:Palmitates; D012333:RNA, Messenger; D014212:Tretinoin; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors